Black soybean hull extract, method for obtaining, and use thereof

ABSTRACT

Methods for preparing black soybean hull extracts and uses thereof. The methods can comprise: (1) extracting crushed black soybean hull with aqueous solution containing enzyme and filtering the extracted liquid; (2) ultrafiltration of the extract liquid with an ultrafiltration membrane; (3) absorbing the ultrafiltration liquid with an absorption resin or an ion exchange resin, and elution with ethanol to obtain purified extract liquid; (4) concentrating the purified extract liquid under vacuum; (5) spray drying the concentrated liquid. The extract can contain: 10-45% of cyanidin, 10-25% of catechin, and 40-80% of OPC, and with a total polyphenol content as high as 70-100%. The extract can be used for preparing drugs or food containing antioxidants, or used for improving metabolic syndrome or eyesight.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a divisional of U.S. Ser. No. 11/830,442, filed Jul.30, 2007, which is a continuation-in-part and claims benefit under 35U.S.C. §120 of International Application No. PCT/CN2006/003397, havingan International Filing Date of Dec. 13, 2006.

TECHNICAL FIELD

This document relates to an extracting method and an extract of anatural product, and in particular, to a method for extractingpolyphenol from black soybean, to black soybean hull extracts obtainedby the method, and to the use of the black soybean hull extracts.

BACKGROUND

Black soybean is also referred to as black bean, lu bean, liao bean, orlingwu bean. According to the theory of traditional Chinese medicine,black material can have an effect on kidney, and black soybean, beingsweet-warm and innocuous, can affect kidney and spleen as well as heartmeridian. Black soybean is thought to have function in tonifying kidneyand improving physique, dehumidification and diuresis, as well asresisting aging and prolonging life. Eating black soybean is believed toimprove skin, benefits look, replenish marrow, enhance strength, andincrease appetite. Researchers have found that black soybean is highlynutritious, and is one of the five kinds of black food which are verypopular in the market.

Researchers have shown that there are plentiful nutritious substances inthe black soybean hull, and that black soybean has high protein contentand low caloric content. Black soybean contains eighteen amino acids,especially the eight amino acids that are necessary for the human body.Black soybean also contains nineteen oleic acids, and the unsaturatedfatty acid content of the black soybean is as high as 80%, with anabsorptivity of over 95%. Black soybean can lower cholesterol content inblood besides meeting people's demand for fat. Generally, black soybeancontains no cholesterol, but rather contains plant sterols, which cannotbe absorbed by the human body. Plant sterols restrict the absorption ofcholesterol by the body, as well as lower the content of cholesterol inblood. The levels of microelements in black soybean such as zinc,copper, magnesium, molybdenum, selenium, and fluorine also are veryhigh. These microelements play an important role in resisting aging ofthe human body, lowering blood viscosity, etc. The black soybean hull isblack in color and contains anthocyanidin, which is a good source ofantioxidants and can eliminate free radicals from the body.Anthocyanidin is thought to have an especially good antioxidation effectin the acid environment of stomach.

Many human diseases are thought to be related to unbalanced freeradicals, including cancer, diabetes, and ophthalmologic diseases. Innormal situations, the generation and elimination of oxygen-derived freeradicals is balanced. When too many oxygen-derived free radicals aregenerated or the antioxidant system is disabled, however, metabolism ofoxygen-derived free radicals in the body will become unbalanced.Polyphenolic compounds are antioxidants, and can be divided into twocategories: (1) monomers of polyphenol, including various flavonoids andchlorogenic acid, and composite polyphenolic compounds containingglycoside; and (2) oligomers or polymers polymerized from monomers,generally referred to as tannin. Anthocyanidin is one example of apolyphenol. See, Zheng et al., “Production and Use of Antioxidants,”published by Chemical Industrial Publishing company.

Chinese Patent Application No. 200510036419.3 discloses a method forpreparing black soybean seed hull anthocyanin comprising the followingsteps: 1) peeling black soybean seed hull as the raw material; 2)grinding black soybean seed hull into fine powder with a super-micropulverizer, and processing the fine powder with a micro-wave machine; 3)extracting the crushed black soybean seed hull anthocyanin with 60-80%ethanol as the solvent; 4) condensing the extracted solution of theblack soybean seed hull anthocyanin obtained in step 3 in a vacuum untilthe concentration of solid substances in the solution is over 40%; 5)degreasing, absorbing, and desorbing the extracted condensed solutionobtained in step 4 with 65-75% ethanol, thus obtaining the purifiedextraction solution of the black soybean seed hull anthocyanin; and 6)condensing the extraction solution obtained in step 5 in a vacuum againuntil the concentration of solid substances is over 60%, and thenfreeze-drying the resulting substance to obtain the black soybean seedhull anthocyanin. The yield is said to increase remarkably when thefineness of the black soybean seed hull is over 320 meshes.

Chinese Patent Application No. 88104657.4 discloses methods forextracting a natural brown pigment, including an extracting method usingwater and an extracting method using ethanol. The extracting methodusing water is as follows: the seed hull of black soybean or brownsoybean is added with 5-15 times of water, and is soaked for 10-24hours, then is refluxed for 1 hour or boiled for 1-2 hours and thenfiltered. The aforesaid steps are repeated three times, and thefiltrates are combined, decompressed and condensed, then 95% of ethanolis added into the condensed solution, and deposits are filtered afterthe condensed solution is placed still for a time. The resultingsolution is then decompressed, concentrated, and dried to obtain thenatural brown pigment. In the extracting method using ethanol, 15%-50%ethanol is used to soak the seed hull, and the other steps are the sameas those in the extracting method using water.

Japanese Patent Application No. 2004-238303 discloses a method forextracting a black soybean seed hull extract composition. The methodincludes the following steps: the black soybean seed hull is extractedin an acid solution with a pH 1-2, then is neutralized until the pHreaches 3.0-4.0, and filtered, then desalted, condensed, and refined.The contents of substances are adjusted through an adding procedure, andthen the resulting substance is dried to be the final product. Accordingto the method, the content of the anthocyanin as the effective componentis over 10% by weight, and the content of the polyphenol is over 50% byweight determined by UV absorbance analysis.

Although the extracted target substances are not the same for the aboveextracting methods, the methods have the same general defects. The yieldobtained by the extracting method using ethanol is relatively high, butthe production cost is also high; the extracting method using water isof low cost, but the yield is also low. Further, the purity of productsobtained by extracting methods using water or ethanol is not high.Therefore, an extracting method by which higher yield and products ofhigher purity can be achieved is desired.

SUMMARY

The present document provides methods for preparing a black soybean hullextract (BSHE) that are cost effective and give an increased yield ascompared to other methods known in the art. The methods provided hereincan comprise:

(1) an extracting step: extracting the crushed black soybean hull withwater solvent containing enzyme and filtering the extracted solution toobtain the filtration solution;

(2) an ultrafiltration membrane filtering step: filtering the filtrationsolution with an ultrafiltration membrane, thereby obtaining anultrafiltration filtrate;

(3) a resin separating step: absorbing the ultrafiltration filtrate withabsorbing resin or ion exchange resin, and eluting the same withethanol, thereby obtaining purified extract solution;

(4) an concentrating step: condensing the purified extract solution invacuum, thereby obtaining a condensed solution; and

(5) a spray-drying step: spray-drying the obtained condensed solution,thereby obtaining BSHE.

According to the preparation method, the water solvent containing enzymecan contain cellulase or pectinase with a concentration of 0.05%-0.2%.

The preparation method further can comprise a step wherein the crushedblack soybean hull is sieved with a screen of 60-80 meshes, wherein inthe extracting step, the extracting temperature is 20-60° C., theextracting time is 1-2 hours, and the extracting steps are repeated 2-3times.

According to the preparation method, in the ultrafiltration membranefiltering step, remaining enzyme and sugar of high molecular weight, aswell as other dregs, can be removed, the diameter of the holes of theultrafiltration membrane can be 0.1-10 um; the molecular weight cut-offcan be 8000-10000 Da; the temperature can be 10-45° C.; the pressure canbe controlled at 2-3 bar; and the flux can be 100-150 L/h.

According to the preparation method, 40-95% ethanol can be used forelution in the resin separating step. The resin can be an absorptionresin of various polarities or an ion exchange resin of variousintensities.

According to the preparation method, the content of the solid substancein the concentrated liquid obtained in the concentrating step can be upto 10%-30%, and the temperature of vacuum concentration can be below 60°C.

The present document further provides the BSHE prepared according to themethods disclosed herein, wherein the BSHE contains: 10-45% cyanidin,10-25% catechin, and 40-80% oligomer proanthocyanidin (OPC), and whereinthe total content of polyphenol is at least 60% (e.g., at least 70%, atleast 80%, or at least 90%).

The present document further provides the use of BSHE prepared accordingto the methods disclosed herein, for preparing antioxidants andpharmaceuticals or foods that can ameliorate metabolic syndrome.

The present document further provides the use of BSHE prepared accordingto the methods disclosed herein, for improving eyesight.

The present document also provides a composition (e.g., a nutritiousfood composition) comprising BSHE, wherein the BSHE contains: 10-45%cyanidin, 10-25% catechin, and 40-80% of OPC, and wherein the totalcontent of polyphenol is at least 60% (e.g., at least 70%, at least 80%,or at least 90%). The composition can be a dietary or food supplement.

This document also features a method for improving eyesight in a subjectin need thereof, the method comprising administering to the subject aneffective amount of the BSHE as described herein.

In addition, this document features a method for treating metabolicsyndrome, the method comprising administering to a subject diagnosedwith metabolic syndrome an effective amount of the BSHE as describedherein.

In another aspect, this document features a method for preparing BSHE,comprising extracting crushed black soybean hull with an aqueoussolution containing an enzyme. The method further can comprise filteringthe extracted liquid to obtain a filtration liquid. The method also canfurther comprise filtering the filtration liquid with an ultrafiltrationmembrane to obtain an ultrafiltration liquid, absorbing theultrafiltration liquid with a resin and then eluting with ethanol toobtain a purified extract liquid, concentrating the purified extractliquid under vacuum to obtain a concentrated liquid, and spray dryingthe concentrated liquid to obtain a BSHE. The ultrafiltration caninclude removing remaining enzyme, large molecule weight sugars, andother dregs; the aperture of the ultrafiltration membrane can be 0.1 μmto 10 μm, the molecular weight cut-off can be 8000 Da to 10000 Da; thetemperature can be 10° C. to 45° C., the pressure can be controlled at 2bar to 3 bar, and the flow rate can be 100 L/h to 150 L/h. The resin canbe an absorption resin or an ion exchange resin. 40% to 95% ethanol canbe used for said eluting. The content of solid substance in theconcentrated liquid produced in the concentrating step can be 10% to30%, and the temperature of the vacuum concentration can be below 60° C.The aqueous solution containing enzyme can contain cellulase orpectinase (e.g., 0.05% to 0.2% cellulase or pectinase). The method canfurther comprise sieving the crushed black soybean hull with a screen of60-80 meshes, wherein the temperature for the extracting step is 20° C.to 60° C., and the extracting is performed for 1 to 2 hours and isrepeated 2 to 3 times.

The advantages of the methods, extracts, and composition provided hereinare as follows:

1. The fineness of the seed hull is 60-80 meshes, and needs not be toofine.

2. Compared with the cost of traditional alcohol solvents, the cost ofthe water solvent added with enzyme is reduced; while subsequentprocessing of the extraction solution obtained through extraction usingwater as the solvent can be conducted without condensation, and is easyto do; and compared with the acid water extraction process, the emissionof effluent is decreased, which is helpful for environmental protection.

3. The content of polyphenolic substances is higher than that obtainedthrough extracting methods using alcohol or water—as high as 80% or even90% or more.

4. The yield using the preparation methods provided herein is higherthan that of extracting methods using water and alcohol.

5. Various effective components can be extracted fully, and all theeffective components can be retained to a great extent.

Unless otherwise defined, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention pertains. Although methods and materialssimilar or equivalent to those described herein can be used to practicethe invention, suitable methods and materials are described below. Allpublications, patent applications, patents, and other referencesmentioned herein are incorporated by reference in their entirety. Incase of conflict, the present specification, including definitions, willcontrol. In addition, the materials, methods, and examples areillustrative only and not intended to be limiting.

The details of one or more embodiments of the invention are set forth inthe accompanying drawings and the description below. Other features,objects, and advantages of the invention will be apparent from thedescription and drawings, and from the claims.

DESCRIPTION OF DRAWINGS

FIG. 1 is a chromatogram of BSHE produced as described in Example 1.

FIG. 2 is a fitted line showing the clearance function of ascorbic acidto hydroxy free radical.

FIG. 3 is a fitted line showing the clearance function of BSHE tohydroxy free radical.

DETAILED DESCRIPTION

Unless otherwise described, the ratio and content referred to in thepresent document is by mass.

The term “black soybean hull extract” (BSHE) in the present documentrefers to compositions extracted from black soybean hull, wherein thetotal content of polyphenol is as high as 90% or more, cyanidin contentis 10-45%, catechin is 10-25%, and OPC is 40-80%. The composition mainlycomprises polyphenolic substance and has functions of antioxidantactivity, improving eyesight, antimetabolic syndrome, etc.

The term “catechin” in the present document refers to catechinpolyphenols, mainly comprising catechin and epicatechin. The term “thetotal content of polyphenol” refers to the total content of variouspolyphenolic substances (which mainly includes cyanidin, catechin, andOPC), and the value of the total content of polyphenol(s) is more thanor equivalent to the total content of cyanidin, catechin, and OPC.

The BSHE extracting methods provided herein are mainly characterized inthat the crushed black soybean hull is extracted with an aqueoussolution containing enzyme as the solvent, and further, in thatseparation steps of ultrafiltration membrane filtration and resinabsorption are employed. The method can comprise one or more of thefollowing steps:

1. Peeling and Crushing Step: The seed hull of black soybean can bepeeled (an existing peeler can be used), and the peeled black soybeanseed hull can be crushed and sieved. A screen of 50-100 meshes can beused for the sieving, e.g., a screen of 60-80 meshes. The number ofmeshes of the screen can affect the quality of the extracts. If too fineof a screen is used (e.g., if the number of meshes is over 100, such as200 or 300 meshes), there may be many impurities in the extract, and ifa too rough of a screen is used (e.g., if the number of the meshes isbelow 50), the yield may be low and it may be hard to get the desiredeffective components. When a screen of 60-80 meshes is used, the desiredresults can be achieved. It will be understood that the peeling step forthe black soybean and the grinding step for the hull can be separatedfrom the extracting step, and can serve as the preparation step for themethods provided herein.

2. Extracting Step: Extraction can be conducted with an aqueous solutioncontaining 0.05-0.2% enzyme as a solvent. The ground (or crushed) blacksoybean hull can be mixed with the solvent in a proportion of 1:10-15 byweight. The mixture can be heated and the temperature kept at 20-60° C.The extracting time can be 1-2 hours, and the extracting can be repeatedtwo or more times (e.g., 2 times, 3 times, 4 times, or more than 4times). The mixture can be filtered after each extracting, and filtratescan be combined. Filtration can be conducted by traditional filteringmethods. Compared with extracting methods using water or ethanol, theyield can be improved or the cost can be reduced by extracting thecrushed black soybean hull with a solvent of water containing enzyme.Compared with extracting methods using acid solution of pH 1-2, such asthat disclosed by Japanese patent application 2004-238303, the methodsprovided herein have low equipment requirements (e.g., no anti-acidprocessing is required), and are environmentally friendly.

In the method provided herein, the selection of enzyme is veryimportant. If the type or dosage of the selected enzyme is improper, theeffective components of the product will be ruined. The inventors havefound that better results are achieved using pectinase or cellulase.

Pectinase (or pectase) mainly comprises pectin lyase, polygalacturonase,pectate lyase, and pectinesterase. Pectinase can effectively decomposethe pectin in plant tissue. Pectic substance is highly esterifiedpolygalacturonic acid. When pectinase acts on a pectic substance, pectinlyase, polygalacturonase, and pectate lyase act directly on theglycosidic linkage inside the molecular chain of the pectic polymer, andthe pectinesterase hydrolyzes polygalacturonate to create more positionsfor polygalacturonase and pectate lyase. Pectinase can be extracted fromplants such as tomato, or from microorganism culture. Pectinase also iscommercially available.

Any suitable concentration of pectinase can be used. A concentrationfrom about 0.05% to about 0.2% (e.g., about 0.075%, about 0.1%, about0.125% about 0.15%, or about 0.175%) can be particularly useful. If theconcentration of pectinase in the aqueous solution is below 0.05%, theyield can be low, and it can be hard to extract fully effectivecomponents. If the concentration of pectinase in the aqueous solution isabove 0.2%, a portion of the desired components may be ruined and thecost will be increased.

Cellulase also can be used. The inventors have observed that cellulasecan be more effective than pectinase, as it can reduce the viscosity ofthe extract, thus facilitating filtration. “Cellulase” refers to thegeneric term of a group of enzymes that can hydrolyze theβ-1,4-glycoside bonds of cellulose to convert cellulose into cellobioseand glucose. Cellulase is not a single enzyme, but rather is amulti-component system of enzymes that have synergetic effects. See, Heet al., “Cellulase and its Current Situation of Application,” Food andFat, 2004, 1:15-18. Sources for cellulose, include, for example, insectsand microbes, such as bacteria, antinomycetes, fungi, etc. Microbialfermentation can be an effective way to produce cellulase on a largescale. Production processes for cellulose include solid fermentation andliquid fermentation. Raw materials for production include, for example,bran, straw powder, waste paper, corn powder, and inorganic salts. See,Qi et al., “Production and Application of Cellulase,” Hebei ChemicalIndustry, 2000, 1:25-26. Cellulase is mainly used in the food industry,forage industry, aquatic product industry, detergent industry, andtextile industry. The extraction methods provided herein can include theuse of commercially available cellulase. The concentration of cellulasein the aqueous solution can be about 0.05 to about 0.2% (e.g., about0.075%, about 0.1%, about 0.125% about 0.15%, or about 0.175%), which issimilar to the amount of pectinase used. Without being bound by aparticular mechanism, the mechanics of applying cellulase are asfollows: cellulase may promote decomposition of cellulose andhemicellulose and dissolution of the plant cellular wall to dissolvemore contents of plant cells. Furthermore, cellulase can degrade largemolecular amylase, which is not easily digested into small molecularmaterials.

3. Ultrafiltration Membrane Filtering Step: The filtrate can be filteredwith an ultrafiltration membrane, thereby obtaining an ultrafiltrationfiltrate. Remaining enzymes, large molecular sugars and other dregs canbe removed during the filtering step. The aperture of theultrafiltration membrane can be from about 0.1 to about 10 μm, themolecular weight cut-off (MWCO) can be from about 8000 to about 10000Da, the temperature can be about 10° C. to about 45° C., the pressurecan be controlled at about 2 to about 3 bar, and the flow rate can beabout 100 to about 150 L/h. Ultrafiltration membranes can becommercially obtained. It is noted that ultrafiltration membranefiltration is not required in the present methods, however, as theseparation method of alcohol deposition also can be used.

4. Resin Separating Step: This step can include adsorbing theultrafiltration filtrate with an adsorption resin or an ion exchangeresin, and eluting the same with ethanol (e.g., about 40% to about 95%ethanol), thereby obtaining a purified extract solution. Variouspolarities of adsorbing resin or various strengths of ion exchange resincan be used, although current adsorbing resins of medium polarity orcation ion exchange resins can be particularly useful.

5. Concentrating Step: The purified extract solution can be concentratedto obtain a condensed solution. By condensation under reduced pressure,the solution can be concentrated until the concentration of solidsubstances in the condensed solution is about 10% to about 30%. Thecondensing temperature can be below 60° C. (e.g., about 50° C. to about55° C.), and the vacuum degree can be about −0.08 MPa. When thetemperature is lower than 40° C., the condensing rate may be too slow,and when the temperature is higher than 60° C., too much product may belost.

6. Drying Step: The obtained condensed solution can be spray dried toobtain the BSHE. Spray drying can be carried out using standard spraydrying equipment. If the method includes ultrafiltration membranefiltering, resin separating, and spray drying, very little product lossmay be achieved. This may help to further increase the yield of BSHE.

BSHE obtained by the methods provided herein typically is highlypurified. The total content of polyphenol in the BSHE may reach 60% orhigher (e.g., at least 70%, at least 80%, or at least 90%), wherein thecontent of cyanidin reaches about 10% to 45%, the content of catechinreaches about 10% to 25%, and the content of OPC reaches about 40% to80%.

It is appreciated that traditional separating methods, for example,methods including condensing, alcohol depositing, and drying steps, maybe applied after extracting the crushed black soybean hull using anaqueous solution containing an enzyme. However, by use of the methodsprovided herein, the yield and the purity may be increased. Further themethods provided herein are simple, and the production of BSHE isfeasible at low cost. In addition, the products contain various kinds ofnutrients, and also can be used in health care compositions.

BSHE obtained by the methods provided herein is rich in polyphenols, andmay be used alone or in combination with other effective materials toproduce a nutritional food composition. Such compositions can be used toprevent or treat (e.g., to improve symptoms) physiological dysfunctionthat is related to high oxidation activity and low demolition of freeradicals, including, for example, metabolic syndrome, eyesight reductionor impairment, and eyestrain. The nutritional composition can be a foodsupplement (e.g., a capsule or pastille, or a filled in micro-sizedcapsule), or can be included in a food (e.g., in alcohol-free softdrinks, milk, juice, pastries, or in teabags). BSHE also can be preparedinto a vegetable pharmaceutical composition. Such a vegetablepharmaceutical composition can be in a form suitable for oraladministration, for example, a pastille, hard or soft capsule, aqueoussolution or syrup, or a form that is suitable for external use. Thedosage of BSHE can be adjusted on demand.

Partially or highly purified polyphenol in an effective dose can beformulated into a pharmaceutical composition with a pharmaceuticallyacceptable carrier, which can be used to treat physiological dysfunctioncaused by oxygen free radicals. An “effective dose” refers to a dose ofpolyphenol sufficient to show a beneficial effect. The effective dosecan be varied, as a person skilled in the art can understand, dependingon the method of administration, the excipient used, and whether otheroptional co-employed therapeutics are used.

The toxicological research of BSHE and the application of the extract inthe aspects of curing eyestrain, eyesight dropping, and metabolicsyndrome also are included in the scope of the present document.Experiments in vitro or in vivo can be used to evaluate theanti-oxidation effects of the nutritional or pharmaceutical compositionsprovided herein. For example, the ability of compositions containingBSHE to reduce or prevent formation of free radicals can be evaluatedvia in vitro or in vivo experiments.

Using an ortho-phenanthroline Fe²⁺ oxidation method to test hydroxylfree radical levels, the inventors observed that BSHE has the effect ofscavenging hydroxyl free radicals, and that its scavenging capability isrelated to the concentration of pharmaceuticals. The scavenging rate ispositively correlated with the concentration, and its IC₅₀ is quite abit lower than the IC₅₀ of ascorbic acid (Vc) under the sameexperimental conditions. The inventors concluded that BSHE has greaterscavenging capabilities than ascorbic acid, and has significantanti-oxidation effect in vitro.

BSHE has shown no toxic effects in a series of toxic experiments, suchas orally acute toxic tests in mice, Ames tests, mouse bone marrow cellmicro-nuclei tests, mouse sperm malformation tests, etc. Thus, BSHE canbe considered as safe and innocuous to the human body.

The effect of BSHE obtained by the methods disclosed herein on the humanbody with respect to eyesight protection and metabolic syndrome wasinvestigated. As described in greater detail below, sixty young peoplewhose far eyesight was degraded and whose age ranged from 7 years to 28years were randomly divided into a test group and a control group. Thosein the test group took BSHE orally at 300 mg/day, and each person in thecontrol group took the same dose of placebo every day. After two months,changes in eye symptoms and eyesight were assessed. Compared to thecontrol group, the BSHE test group showed significant improvement in eyesymptoms and distance eyesight. It was concluded that BSHE is capable ofreducing eyestrain symptoms and improving distance eyesight of earlynear-sighted people and lightly near-sighted people.

Sixty-two patients diagnosed with metabolic syndrome whose ages rangedfrom 32 years to 81 years were randomly divided into a test group and acontrol group. Besides their daily regular treatment, those in the testgroup further took BSHE orally at 300 mg per day. Those in the controlgroup took the same dose of placebo. After two months, changes inweight, blood lipid, blood pressure, and blood sugar on an empty stomachand two hours after a meal were measured. It was observed that weight,blood pressure, blood lipid, and blood sugar were significantlydecreased in the test group. It thus can be concluded that adding BSHEto regular treatment may help drugs to treat or cure metabolic syndrome,and may be advantageous to prevent the occurrence and aggravation ofdiabetes and cardiovascular diseases caused by metabolic syndrome.

The invention will be further described in the following examples, whichdo not limit the scope of the invention described in the claims.

EXAMPLES Example 1 BSHE Preparation 1

One thousand g crushed black soybean hull (passing through a sieve with60 meshes) as the raw material was extracted with 15 L of an aqueoussolvent solution containing 0.08% pectase at 40° C. for two hours, thenfiltered. The extraction process was conducted twice and during thesecond extraction process, 10 L aqueous solution with the same pectaseconcentration was added. The extraction liquid from the two processeswas combined (27 L) and then filtered through an ultrafiltrationmembrane until the resulting effluent was about 20 to 25 L. The effluentwas subsequently resin separated and purified (at a flow rate of 20 to40 L/h), and then eluted with 40-95% ethanol. The ethanol-eluting liquidwas combined and then concentrated until the solid substance in theconcentrated solution was equal to or more than 30%. Thirty-five g BSHEwas obtained, and the yield was 3.5%.

The resulting BSHE product contained 32.54% cyanidin, 15.63% catechin,and 50.01% OPC, and the total content of polyphenol was up to 98.18% ormore. The components were measured using a high efficiency liquid phasechromatogram (HPLC), and the chromatogram is shown in FIG. 1. In thechromatogram, absorbing peak 1 corresponds to cyanidin, absorbing peak 2corresponds to catechin, absorbing peak 4 corresponds to epicatechin,absorbing peak 3 corresponds to the dimer of OPC, and absorbing peak 5corresponds to the trimer of OPC.

The condition of the measurement of chromatogram were:

-   -   1. the chromatogram column: ODS C18, 5 μm, 250 mm×4.6 mm    -   2. flow phase A: 9% acetonitrile, 0.1% glacial acetic acid        (aqueous solution) flow phase B: 40% acetonitrile (aqueous        solution)

time (min) flow phase A flow phase B 0.01 94% 6% 10.00 94% 6% 50.00 0100% 55.00 0 100% 60.00 94% 6%

-   -   3. flow rate: 0.8 ml/min    -   4. determination wave length: 280 nm    -   5. temperature of the column: 40° C.

Example 2 BSHE Preparation 2

One thousand g crushed black soybean hull (passing through a sieve with90 meshes) as the raw material was extracted at 55° C. with 15 L of anaqueous solvent solution containing 0.2% cellulose. The temperature wasmaintained for two hours, and the extract was filtered. The extractionprocess was conducted twice, and during the second extraction process,10 L of aqueous solution of the enzyme at the same concentration wasadded. The remaining steps were as described in Example 1. Forty-eight gof BSHE were obtained, and the yield was 4.8%. The resulting BSHEproduct contained: 12.52% cyanidin, 13.50% catechin, 54.01% OPC, and thetotal content of polyphenol was up to 80.03%.

Example 3 BSHE Preparation 3

The preparation steps of Example 3 were the same as those of Example 1,except that the aqueous solution contained 0.08% cellulase instead of0.08% pectase. Thirty-eight g of BSHE were obtained, and the yield was3.8%. The resulting BSHE product contained: 27.43% cyanidin, 11.25%catechin, and 54.12% OPC, and the total content of polyphenol was up to92.80%.

Example 4 BSHE Preparation 4

One thousand g crushed Black Soybean Hull (passing through a sieve with70 meshes) as the raw material was extracted at 50° C. with 15 L of anaqueous solvent solution containing 0.1% cellulose. The temperature wasmaintained for 1.5 hours, and the extract was filtered. The extractionprocess was conducted twice, and during the second extraction process,10 L of the aqueous enzyme solution was added. The remaining steps werethe same as those described in Example 1. Fifty-five g of BSHE wasobtained, and the yield was 5.5%. The resulting BSHE product contained:13.52% cyaniding, 11.70% catechin, and 52.01% OPC, and the total contentof polyphenol was up to 77.23%.

Example 5 Comparative BSHE Preparation Using 50% Ethanol

One thousand g crushed black soybean hull (passing through a sieve with60 meshes) as the raw material was extracted at 40° C. with 15 L of a50% ethanol solution as a solvent. The temperature was maintained for 2hours, and the extract was filtered. The extraction process wasconducted twice, and during the second extraction process, 10 L of a 50%ethanol solution was added. The remaining steps were the same as thosedescribed in Example 1. Three g of BSHE were obtained, and the yield was3%. The resulting BSHE product contained: 9.63% cyanidin, 7.53%catechin, and 30.82% OPC, and the total content of polyphenol was47.98%.

Example 6 Comparative BSHE Preparation Using Water

One thousand g crushed black soybean hull (passing through a sieve with60 meshes) as the raw material was extracted with 15 L water as asolvent, and circumfluence was conducted twice. The extraction processwas conducted twice. The filtrates from the two processes were combinedand concentrated. The remaining steps were the same as those describedin Example 1. One and a half g of BSHE were obtained, and the yield was1.5%. The resulting BSHE product contained: 5.61% cyanidin, 10.83%catechin, and 35.93% OPC, and the total content of polyphenol was52.37%.

It can be seen that the yield using the aqueous solution containing theenzyme as the extraction agent was higher than the yields obtained usingethanol or water as the extraction agent. The total content ofpolyphenol using the aqueous enzyme solution also was higher than thepolyphenol content obtained using the ethanol or water, even though theother conditions were the same.

Example 7 Evaluation of BSHE Toxicity

Based on the mice acute toxicity test, the maximum tolerable dosage ofBSHE is more than 8 g/kg body weight. When the highest dosage of thesample was 100 times the recommended human dosage (300 mg/kg), theresults did not indicate any mutagenicity based on the Ames test, mousemarrow cell microkernel test, and mouse semen abnormality test.Consequentially, it is believed that the extract of black soybean issafe for humans in terms of toxicology.

Example 8 Anti-Oxidation Effects of BSHE In Vitro

Materials and methods-test drug and reagent: Extract of black soybeanhull A (containing cyanidin 12.52%, catechin 13.50%, and OPC 54.01%).Orthophenanthroline, hydrogen peroxide, ascorbic acid (VC), andFeSO₄.7H₂O were all analytical pure reagents made in China.

Materials and Methods-instruments: An LSY constant temperature waterbath was obtained from a Beijing medical instrument manufacturer, and aUV-2000 spectrophotometer was from Tainike, Shanghai Instrument Co.,Ltd.).

Test method-reagent preparation: Reagents included 0.1% H₂O₂ (V/V), a 5mmol/L anhydrous ethanol solution of orthophenanthroline, 0.75 mmol/LFeSO₄, a 0.2 mmol/L phosphoric acid buffer solution (PBS; pH=7.4), BSHEA, prepared with double distilled water and diluted to concentrations of0.5, 0.25, 0.125, and 0.0625 mg/ml, and ascorbic acid, prepared atconcentrations of 4, 2, 1, and 0.5 mg/ml with double distilled water.

Test method-measuring hydroxyl free radicals: The clearing of hydroxylfree radicals was tested using an orthophenanthrolin Fe²⁺ oxidationmethod to further evaluate the anti-oxidation capacity of BSHE A.

(1) 2 ml PBS, the test drug at the concentrations listed above, and 1 mlascorbic acid were sequentially added to a disposable 10 ml polyethylenetest tube and shaken. 0.35 ml of 5 mmol/L orthophenanthroline solutionand 1 ml 0.75 mmol/L FeSO₄ were added and mixed fully. 1 ml of 0.1% H₂O₂was added, and the tube was vibrated for 1 minute. The tube wasincubated at 37° C. for 60 minutes, and the absorbency value Aa of thereaction system at λ=510 mm was measured using a UV-2000spectrophotometer.

(2) The 1 ml of test drug in (1) was replaced with 1 ml deionized water,while the other conditions were the same as in (1). The absorbency valueAb was measured.

(3) The 1 ml H₂O₂ in (2) was replaced with 1 ml deionized water, whilethe other conditions were the same as in (2). The absorbency value Acwas measured.

(4) The clearance rate (d) of .OH by the test drug and the positivecomparison was calculated according to the following equation:

$d = {\left( {1 - \frac{A_{a} - A_{c}}{A_{b} - A_{c}}} \right) \times 100\%}$

The total volume of reaction was 5.35 ml. The final concentrations ofascorbic acid thus were 0.748 mg/ml, 0.374 mg/ml, 0.187 mg/ml, and 0.093mg/ml, while the final concentrations of BSHE A were 0.093 mg/ml, 0.047mg/ml, 0.023 mg/ml, and 0.012 mg/ml. Five samples were tested in eachgroup. The clearance rate of .OH was calculated individually and thenaverage values were calculated. The results are shown in Table 1.

TABLE 1 Clearance effects of ascorbic acid on hydroxyl free radical (n =5) Final concentrations (mg/ml) Clearance rate of •OH (d) 0.748 91.29 ±3.26 0.374 50.32 ± 4.29 0.187  4.21 ± 1.32 0.093 0.212 ± 2.47

In this test, when testing the clearance effect of BSHE on hydroxyl freeradicals, ascorbic acid, the known clearance agent of .OH, was used as apositive comparison to verify the reliability of the method. Its fittedline was Y=−14.6996+146.1028X, and the regression coefficient was 0.983.The IC₅₀ was 0.4014 mg/ml, as shown in FIG. 2.

TABLE 2 Clearance effect of BSHE A on hydroxyl free radicals (n = 6)Final concentrations (mg/ml) Clearance ratio of •OH (d) 0.093 89.87 ±11.07 0.047 39.52 ± 3.33  0.023 14.96 ± 1.79  0.012 3.39 ± 2.00

The fitted line was Y=−9.5012+1060.1879X, with a regression coefficientof 0.9999. The IC₅₀ was 0.0456 mg/ml, as shown in FIG. 3.

In the reaction system of this test, the aqueous solution of BSHE cancombine with the hydroxyl free radicals produced from the Fentonreaction, reducing the oxidation of Fe²⁺ due to the free hydroxylradicals. The clearance effects of BSHE on free hydroxyl radicals wastested through measurement of the absorbency value of the reactionsystem. A portion of Fe²⁺ is oxidized to Fe³⁺ by hydroxyl free radicals,both of which coexist in the reaction system for a short time.

The drug may enhance the absorbency value of the reaction system bycombining with hydroxyl free radicals to reduce the oxidation on Fe²⁺,increasing the concentration of the combination (conjugation substance)of orthophenanthroline-Fe²⁺ in the reaction system, and showing theclearance effect of the drug on .OH.

BSHE may combine with hydroxyl free radicals via the mechanism describedabove, reducing the oxidation of Fe²⁺ due to hydroxyl free radicals,thereby showing the effect of eliminating hydroxyl free radicals. Therewas an obvious amount-effect relationship between the eliminatingcapacity of BSHE and the concentration of the drug, where theelimination ratio of BSHE was positively correlated with theconcentration of the drug, and the IC₅₀ was 0.0456 mg/ml. On the otherhand, under identical test conditions, the IC₅₀ of ascorbic acid (Vc)was 0.4014 mg/ml, which indicates that the clearance capacity of BSHEfor hydroxyl free radicals is stronger than that of ascorbic acid, andthat BSHE has significant antioxidation effects in vitro.

Example 9 In Vivo Effects of BSHE on Eyesight

Sixty volunteers with reduced distant vision, ranging from 7 to 28 yearsof age, were selected. The recruiting criteria for tested personsincluded: 4.0<=naked eye distance vision<5.0; naked eye near vision≧5.0;it was considered pseudomyopia or myopia if diopter was ≦3D aftermydriatic test. Exclusion criterion of the tested persons included:persons of an age not from 7 to 28 years; persons with infective eyediseases and other eye diseases such as corneal nebula, keratoconus, orcataracts; persons with eyeground diseases or having cardiovasculardiseases, brain vascular diseases, diabetes, or serious diseases of theliver, kidney, or hematogenic system; persons with psychopathy; personswho had been using other relevant drugs or therapeutics for an extendedtime and who were not currently pausing from their therapeutics; andpersons who failed to eat according to regulation, failed to judge thefunction, or did not have complete and accurate judgment of function andsafety.

Test design and requirements of grouping: Comparison within each groupand between groups were used in this test. The tested persons wererandomly divided into a test group and a control group with 30individuals in each group, according to eyesight level.

Dosage and usage: In the test group, each person orally took three 100mg doses of BSHE per day. In the control group, the placebo wasadministrated at the same dosage. The test duration was 8 weeks.

Observation parameters: Observations monitored included self-sensedsymptoms of eyes (such as eye dryness, blurred vision, and tired eyes),as well as changes in distant vision. A determination of effectivenesswas set at an improvement in distant vision of naked eyes≧2 showing thedrug as effective.

Statistical method: At test was used for statistical evaluation ofeyesight changes, a paired t test was used for comparison informationwithin the same group, and a grouped t test was used for comparison ofaverages between groups.

Results: 60 persons with nearsightedness were observed. The test groupincluded 17 males and 13 females ranging from 8 to 28 years of age, withan average age of 14.32 years. The control group included 14 males and16 females ranging from 7 to 28 years of age, with an average age of14.40 years.

After taking BSHE, for the tested persons, the major uncomfortablesymptoms of eyes were significantly improved. The improvement rate ofeye dryness, blurred vision and eye tiredness were 60.00%, 56.00% and62.96%, respectively, each of which was statistically different ascompared with the control group (P<0.05). After taking BSHE, there wasan apparent improvement in vision within the test group, with astatistically significant difference from vision before testing(P<0.01). The total efficiency ratio was up to 63.33%, and as comparedwith the control group there was a statistically significant difference(P<0.01), as indicated in Table 3 and Table 4.

TABLE 3 Comparison of changes in distant vision of the naked eye beforeand after taking the drug ( x ± s, n = 60) Before After Groupadministration administration Difference Test group 4.55 ± 0.21 4.66 ±0.25 0.60 ± 0.08** Control group 4.56 ± 0.23 4.61 ± 0.22 0.03 ± 0.03ΔΔ**comparison of the test group itself before and after administration; P< 0.01 ΔΔcomparison between groups before and after administration: P <0.01

TABLE 4 Comparison of improvement ratio of distant vision for nakeddouble-eyes between before administration and after administration ( x ±s, n = 60) Group Efficacy Inefficacy Total effect ratio Test group 38 2263.33 Control group 2 58  3.33ΔΔ ΔΔcomparison in groups between groupsbefore and after administration: P < 0.01

Thus, it was observed that youths with myopia obtained significantimprovement in terms of both the naked eye distant vision and theuncomfortable symptoms of eyes after orally taking BSHE for 8 weeks.Vision tiredness involves a variety of symptoms that are mainlypresented as the discomfort of eyes that is caused by longtime or excesseye usage without timely and effective relaxation. There are twopossible inducements. One is that when eyeballs are in a searchingand/or staring situation for a long period of time, the metabolism inextra ocular muscles and ciliary muscles is increased, causing increasedaccumulation of metabolism waste (including oxygen free radicals), suchthat muscle cell structure is damaged and functions are degraded.Another inducement is that visual cells are excessively depleted whilethe required nutriments are deficiently supplied, such that the recoverytime for the macula and retina are extended. It has been observed thatanthocyanin is capable of effectively improving the recovery time formacula of human eyes, especially for the identification of middle andhigh spatial numbers in a dark environment. Consequently, anthocyanidinis considered to be capable of increasing the blood current ofmicrocirculation on the eyeground, speeding the substance metabolismexchange and enhancing the protection effect on the capillary vessel,thus has the effect of improving the recovery time of the macula andnight vision. This could be related to the fact that anthocyanidin canpromote the regeneration of the rhodopsin of retinal rod cells. Inaddition, anthocyanidin is an effective antioxidant that can clear thetoxic oxygen free radical in vivo so as to protect histiocytes. Suchpharmacological actions of anthocyanidin may be the basis of the visionprotection capabilities of BSHE containing plenty of anthocyanidin.

Example 10 Anti-Metabolic Syndrome Effects of BSHE

Tested subjects: Based on the diagnosis criteria (see Table 5) proposedin 2001 by the Adult Treatment Group III (ATP III) of the U.S. NationalCholesterin Treatment Education Program (NCEP), the subjects wereselected from volunteers. Sixty-two cases were selected as satisfyingthe criteria, and were randomly divided into a treatment group and acontrol group. The treatment group included 18 males and 13 females,ranging from 33 to 81 years of age (the average age was 54.0 years), andthe control group included 15 males and 16 females ranging from 32 to 79years of age (the average age was 53.5 years).

TABLE 5 Diagnosis criteria metabolic syndrome Diagnosis criteria(require meeting any 3 of the following 5) Reference critical pointsWaistline Male: 102 cm; Female: ≧88 cm Triglyceride levels ≧1.67 mmol/Lor taking medication for reducing triglyceride High density Male: <1.04mmol/L; Female: <1.29 mmol/L lipoprotein levels or taking medication forincreasing high density lipid protein Blood pressure Systolic pressure≧130 mmHg or diastolic pressure ≧85 mmHg, or taking medication forreducing blood pressure Blood sugar under limosis ≧6.1 mmol/L or takingmedication for reducing blood sugar

Test method: Subjects in the treatment group took oral BSHE 3 times/dayat a dose of 100 mg/time, while patients in the control group were givena placebo at the same dosage. The test duration was 8 weeks.

Observation Parameters:

1) Body weight (kg) was measured in the early morning under conditionsof limosis. Defecation and emiction also were measured beforeadministration of drug or placebo and after administration for 8 weeks.

2) Blood lipid change (total cholesterin and triglyceride) were measuredbefore and after the 8 week administration.

3) Changes in blood pressure were measured before taking and after the 8week administration.

4) Blood sugar changes at limosis and 2 hours after a meal were measuredbefore and after the 8 week administration.

Results: Body mass upon admission was 62.2±8.3 kg in the treatmentgroup, and 61.8±7.8 kg in the control group, with no obvious differencetherebetween (P>0.05). After administration of drug or placebo for 8weeks, however, body mass in the treatment group was 58.4±7.5 kg, whichwas significantly decreased as compared with body mass before treatment(P<0.05). In contrast, body mass in the control group was 60.1±7.6 kg,with no significant difference from that before treatment (P>0.05).

Total cholesterin and triglyceride were significantly reduced afteradministration in both the treatment group and the control group(P<0.01; P<0.05, respectively). There was a significant difference(P<0.01) in reduced levels of total cholesterin between the treatmentand control groups after administration, while there was no significantdifference in levels of triglyceride between the two groups (P>0.05).See, Table 6.

TABLE 6 Changes in blood lipid of patients with metabolic syndromebefore and after administration ( x ± s, n = 31) Total cholesterin(mmol/L) Triglyceride (mmol/L) Before After P Before After Padministration administration value administration administration valueTreatment 7.0 ± 1.0 4.2 ± 0.9ΔΔ <0.01 2.5 ± 1.7 1.4 ± 1.0 <0.05 groupControl 6.8 ± 1.1 5.8 ± 0.9 <0.01 2.5 ± 1.6 1.6 ± 0.9 <0.05 group ΔΔcomparison between groups before and after administration. P < 0.01

Before taking medicine, subjects having normal blood pressure within thetreatment and control groups numbered 16 cases (51.6%) and 17 cases(54.8%), respectively, with no obvious difference therebetween. Afteradministration, however, the number of subjects with normal bloodpressure in the treatment and control groups were 22 cases (70.9%) and19 cases (61.3%), respectively, which was significantly different ascompared with each other (P<0.05).

Blood sugars measured under conditions of limosis and 2 hours aftereating were significantly decreased in both the treatment and controlgroups. For the decreased amount of blood sugar measured 2 hours afterdinner, there was a statistically significant difference between thetreatment and control groups (P<0.01), while there was no significantdifference between the two groups for the decreased amount of bloodsugar under limosis (P>0.05). See Table 7.

TABLE 7 Changes in blood sugar of patients with metabolic syndromebefore and after administration ( x ± s, n = 31) Blood sugar 2 hoursafter eating Blood sugar at limosis (mmol/L) (mmol/L) Before After PBefore After P administration administration value administrationadministration value Treatment 11.7 ± 2.3 7.3 ± 1.0 <0.01 16.0 ± 2.911.1 ± 1.5ΔΔ <0.01 group Control 10.6 ± 1.5 7.9 ± 0.9 <0.05 14.8 ± 1.612.9 ± 0.9 <0.05 group ΔΔ Comparison between groups before and afteradministration: P < 0.01

Based on the above, it has been observed that taking BSHE in addition tonormal treatment can help with treatment of metabolic syndrome,contributing to reduction in levels of body weight, blood pressure,blood lipid, and blood sugar, such that diabetes and cardiovasculardiseases caused by metabolic syndrome can be prevented from occurringand developing. BSHE contains high active substance of anthocyanidins,which can provide antioxidant effects, reducing free radicals anddiminishing inflammation. BSHE thus can be used for, e.g., weight loss,blood lipid reduction, and prevention of cardiovascular diseases.

Other Embodiments

It is to be understood that while the invention has been described inconjunction with the detailed description thereof, the foregoingdescription is intended to illustrate and not limit the scope of theinvention, which is defined by the scope of the appended claims. Otheraspects, advantages, and modifications are within the scope of thefollowing claims.

1. A method used for preparing black soybean hull extract (BSHE),comprising: (1) extracting crushed black soybean hull with an aqueoussolution containing cellulase or pectinase of 0.05-0.2%, and filteringthe extracted liquid to obtain a filtration liquid; (2) filtering thefiltration liquid with an ultrafiltration membrane to obtain anultrafiltration liquid, wherein the molecular weight cut-off of theultrafiltration membrane is 8000-10000 Da; (3) absorbing theultrafiltration liquid with an absorption or ion exchange resin and theneluting with ethanol to obtain a purified extract liquid; (4)concentrating the purified extract liquid under vacuum to obtain aconcentrated liquid; (5) spray drying the concentrated liquid to obtainthe BSHE.
 2. The method of claim 1, further comprising the step of,prior to step (1), sieving the crushed black soybean hull with a screenof 60-80 meshes, wherein in the extracting, step the temperature is20-60° C., and the extracting is performed for 1-2 hours and repeated2-3 times.
 3. The method of claim 2, wherein the filtering step withultrafiltration membrane includes removing the remaining enzyme, largemolecule weight sugar, and other dregs, and wherein the aperture of theultrafiltration membrane is 0.1-10 μm, the temperature is 10-45° C., thepressure is controlled at 2-3 bar, and the flow rate is 100-150 L/h. 4.The method of claim 3, wherein said ethanol in step (3) is 40-95%ethanol.
 5. The method of claim 4, wherein said concentrating of step(4) produces a purified extract liquid having a solid content of about10% to about 30%.